Characterization of a new megabirnavirus that confers hypovirulence with the aid of a co-infecting partitivirus to the host fungus, Rosellinia necatrix

• A new fungal virus Rosellinia necatrix megabirnavirus 2 (RnMBV2) was characterized.
• RnMBV2 resembles other megabirnaviruses (RnMBV1, SsMBV1) in genetic organization.
• RnMBV2 is phylogenetically closer to SsMBV1 than to R. necatrix-infecting RnMBV1.
• Accumulation level of RnPV1 was increased by co-infection with both segments of RnMBV2 in R. necatrix.
• Symptoms in R. necatrix were induced only by co-infection with the two viruses.

A new virus termed Rosellinia necatrix megabirnavirus 2 (RnMBV2) was molecularly and biologically characterized. RnMBV2 was originally harbored in isolate W8 of R. necatrix co-infected with the previously reported virus Rosellinia necatrix partitivirus 1 (RnPV1). RnMBV2 has molecular features similar and different from precedent megabirnaviruses, Rosellinia necatrix megabirnavirus 1 (RnMBV1) and Sclerotinia sclerotiorum megabirnavirus 1 (SsMBV1). The two genomic segments of RnMBV2 (9.0-kbp dsRNA1 and 8.0-kbp dsRNA2) each possess two open reading frames (ORF1 and 2 on dsRNA1 and ORF3 and 4 on dsRNA2), with a well conserved 5′-long untranslated region (UTR) of 1.7–1.8 kb between the segments, and relatively short 3′-UTR. The RnMBV2 dsRNA1-coded capsid protein (CP) and RNA-dependent RNA polymerase (RdRp) show higher sequence identity to those of SsMBV1 than to those of RnMBV1, whereas the RnMBV2 ORF3-coded protein is more closely related to the counterpart of RnMBV1. No significant amino acid sequence similarity was detected among ORF4-coded sequences of the three megabirnaviruses. Virion transfection and co-culturing allowed for single and double infection of mycelial incompatible isolates W37 and W97 by RnMBV2 and/or RnPV1. Their comparative analyses showed RnMBV2 to be able to confer hypovirulence with the aid of a co-infecting RnPV1, while the individual viruses exhibited asymptomatic infections. Interestingly, RnPV1 accumulation appeared to be increased in co-infected fungal strain with two segments of RnMBV2 relative to singly infected fungal strains. Furthermore, the dispensability of RnMBV2 dsRNA2 was demonstrated to be similar to that of the other two megabirnaviruses.